The long-range goal of this proposal is to understand the molecular mechanism by which estrogen regulates prolactin (PRL) gene expression. PRL synthesis is regulated by a number of factors, including estrogen. The binding sites for these regulators are positioned at varying distances from the PRL prom-. In the case of estrogen, the estrogen response element is separated from the promoter by over 1800 base pairs (bp) of DNA. The arrangement of the PRL gene provides a unique situation in which to study long-range transcriptional activation by estrogen. Since both the estrogen receptor (ER) and the tissue-specific pituitary factor one (Pit-1) are required for PRL expression, we wish to determine whether these two proteins are in diet physical contact with each other, thus mediating long-range transcriptional activation of the PRL gene. Thus, we would like to investigate specific protein-protein interactions between the ER and Pit-1. First, the ER-Pit-1 interaction will be analyzed using a Pit-1-protein affinity column in a protein-protein interaction assay. Second, ER will be crosslinked to Pit-1 by chemical crosslinking agents such as glutaraldehyde and bismaleimidohexane and photochemically by UV induction. The crosslinked complexes will be analyzed by standard biochemical and molecular biological techniques. We will address the functional relevance of the ER Pit-1 interaction by demonstrating that the hypothesized interaction between the ER and Pit-1 occurs in vivo. We will use an in vivo UV crosslinking assay to directly measure Pit-1 binding to ER and ER-Pit-1 binding to DNA. The results will provide information regarding the mechanism by which ER and Pit-1 cooperate in regulating the PRL gene.